ESTABLISHING EFFICIENT PROTOPLAST GENOME EDITING SYSTEMS USING CRISPR/CAS9 FOR MAIZE BREEDING AND IMPROVEMENT

Abstract

Amid the growing concerns over global food security, the use of gene editing technology in crop improvement is gaining traction as a promising solution. While this technology has been widely used to modify specific traits in crops, its potential to overcome breeding barriers remains largely unexplored. It is extremely challenging to crossbreed tropical maize and temperate maize under the long-day photoperiod of a temperate environment due to their differences in flowering time. This breeding barrier prevents the integration of tropical maize into temperate breeding programs, greatly limiting genetic diversity. Leveraging the power of the CRISPR/Cas9 system, this study is ultimately focused on three regulators of flowering time, ZmCCT9, ZmCCT10, and ZmRap2.7, to induce earlier flowering in tropical maize, aligning its timing with temperate maize. Since traditional plant transformation techniques are time-consuming and labor-intensive, this study has developed a maize protoplast CRISPR/Cas9 system to rapidly validate the activity of guide RNAs. Additionally, nine gRNAs targeting ZmCCT9, ZmCCT10, and ZmRap2.7 were designed, and their editing efficiencies were evaluated using the protoplast gene editing system.